The present invention relates to a non-invasive method for detecting and monitoring apoptosis. In particular, the present invention relates to a method for detecting apoptosis using ultrasound imaging.
Ultrasound imaging is one of the commonest radiological modalities presently used in clinical medicine. It is used to image the developing fetus, to image organs and vascular components, and to image tumours. Recently, high frequency ultrasound based systems have been developed to produce high resolution images of biological specimens such as spheroids or human tissues in vivo.
High frequency ultrasound imaging has been used to investigate the living and hypoxic regions of cell spheroids indicating the ability of this imaging modality to differentiate different types of cellular morphologies (Sherar et al., Ultrasound backscatter microscopy images the internal structure of living tumour spheroids, Nature 1987; 330: 493-495; Bxc3xa9rubxc3xa9 et al., Use of a high frequency ultrasound microscope to image the action of 2-nitroimidazoles in multicellular spheroids, Br J Cancer,1992; 65: 633-640).
Apoptosis is one mechanism by which biological cells undergo cell death. It plays a significant role in both normal and disease-related biological processes (Hockenbery, D. Defining apoptosis. Am J Pathol, 1995; 146: 16-19; Majno G., Joris I. Apoptosis, oncosis, and necrosis. Am. J Pathol, 1995; 146: 3-15; Fraser A., Evan G. A view to a kill. Cell,1996;85: 781-784). In addition, cells undergo apoptosis in response to a variety of stresses including chemotherapy, radiation therapy, photodynamic therapy and heat. It is useful in both experimental and clinical applications to know whether cells are undergoing apoptosis. This is currently determined by taking samples of the cells or tissues of interest and observing, using histological and DNA measurement methods, whether the cells exhibit the morphological changes that are indicative of apoptosis. These changes include membrane blebbing, DNA condensation and DNA fragmentation. However, these methods are not only invasive, but also time-consuming, requiring processing of a cell or tissue sample before data relating to apoptosis can be obtained.
The ability to differentiate apoptotic cells from living or otherwise dead cells non-invasively in-vitro and in vivo would potentiate clinical diagnoses, the understanding of disease processes and normal biological processes that involve apoptosis, and provide a more efficient way of studying apoptosis in response to therapeutic agents.
Accordingly, the present invention provides a non-invasive method of monitoring apoptosis in cell culture, ex-vivo tissues and in-vivo tissues using high frequency ultrasound imaging, which comprises the steps of
1.) imaging a selected site of the cell culture or tissues using high frequency (above 20 MHz) ultrasound imaging (before image);
2.) exposing the selected site to an apoptosis-inducing stress;
3.) imaging the selected site or a portion thereof, using ultrasound imaging at subsequent timed intervals (after image(s));
4.) measuring the signal amplitude of a region of interest of the selected site in the before and after images;
5.) comparing the signal amplitude measurements for the regions of interest in the before and after images and determining whether the after image regions. exhibit an increase in amplitude as compared to the before image regions which is an indication that apoptosis has begun; and
6.) measuring the change in the frequency spectrum of the radiofrequency ultrasound backscatter signal in the region of interest in the before and after images and confirming that apoptosis has begun when the slope of the frequency spectrum has increased.
In another aspect of the invention, further confirmation of the occurrence of apoptosis is achieved by the step of calculating the average scatterer size in the region of interest by applying an ultrasound scattering model to the radiofrequency ultrasound signals from the regions of interest. Apoptosis is confirmed when the average scatterer size has reduced significantly according to this analysis. The decrease in scatterer size reflects the fragmentation of nuclear material that occurs during apoptosis.
More simply, the method of the invention is to use ultrasound imaging to monitor and measure the apoptotic process in cell culture, ex-vivo tissues and in-vivo tissues using the three-step process of:
1) Imaging of the treated sample or region of tissue before and during treatment and/or imaging treated and untreated regions in the sample or tissue;
2) Measurement of the change in signal amplitude or intensity in the treated and untreated samples or regions of tissue; and
3) Measurement of the change in frequency spectrum of ultrasound scatter from the treated and untreated regions.
An optional fourth step to this process is:
4) Calculation of average scatterer size from the frequency spectra collected.
In essence, the present invention offers a new use of ultrasound imaging to monitor the process of apoptosis. We have discovered that the processes of nuclear condensation and fragmentation that are indicative of apoptosis result in an approximately 3-6-fold increase in the amplitude of ultrasound scattered from cells in culture, ex-vivo tissues and in-vivo tissues as compared to normal cells not undergoing apoptosis. Also, we have discovered that the frequency spectrum of the ultrasound scattered from biological samples and tissues containing cells undergoing apoptosis is different from untreated samples not undergoing apoptosis. The specific subcellular features which permit the apoptotic phenomenon to be visualized have been investigated and are shown to be related to the changes in the cellular nuclear material cells undergo during apoptosis.
Apoptosis is indicated in the sample or region of tissue if both the signal intensity increases and the slope of the frequency spectrum increases. Further confirmation is indicated by a decrease in the average scatterer size in the treated regions. The regions of tissue that satisfy these criteria could be colour coded on the original ultrasound image, for example. There are various methods known in the art for calculating the signal amplitude, slopes of the frequency spectra and the average scatterer size parameters, and any of these may be used, with the choice being one that a person skilled in the art can select readily.
The present method is a non-invasive method utilizing high frequency ultrasound imaging to detect and monitor apoptosis in cells or tissues, in vitro, in vivo or ex vivo. As will be appreciated by those of skill in the art, the term xe2x80x9chigh frequency ultrasound imagingxe2x80x9d is meant to refer to ultrasound imaging at frequencies of greater than 20 MHz. The method involves taking high frequency ultrasound images of the cells or tissues of interest prior to the application of an apoptosis-inducing stress, herein referred to as a xe2x80x9cbefore imagexe2x80x9d, as well as taking a high frequency ultrasound image following application of the stress, referred to herein as an xe2x80x9cafter imagexe2x80x9d. Alternatively, high frequency ultrasound images of treated and untreated regions of the sample or tissue can simultaneously be taken. In this case, the ultrasound image of the untreated region would be equivalent to the xe2x80x9cbefore imagexe2x80x9d and the ultrasound image of the treated region would be equivalent to the xe2x80x9cafter imagexe2x80x9d. The terms xe2x80x9cbefore imagexe2x80x9d and xe2x80x9cafter imagexe2x80x9d as used herein encompass both of the foregoing alternatives.
xe2x80x9cApoptosis-inducing stressxe2x80x9d, as referred to herein, is meant to encompass any stress which will result in the initiation of apoptosis. Examples of apoptosis-inducing stresses include chemotherapeutic agents, drugs, photodynamic therapy, chemical modifiers aimed at protecting tissues from radiations such as sunscreens, radiations including X-rays, gamma rays and ultraviolet radiations, oxygen and/or nutrient deprivation that can occur after organ removal for transplantation for example, and the activation of genes that can initiate an apoptotic response as well as aging and developmental processes. Accordingly, the term xe2x80x9capoptosis-inducing stressxe2x80x9d is also meant to encompass biological events that occur normally in tissues to induce apoptosis.
The quantitative part of the method involves either obtaining the radiofrequency signal and measuring the amplitude, which is the square root of intensity, averaged over a region of interest or if radiofrequency data is not available from the ultrasound machine, using a calibration curve from the ultrasound machine manufacturer to convert the final machine signal into an average radiofrequency amplitude over the region of interest. In either case, the signal amplitude can be measured over the region exposed to the stress and over control untreated regions at any timepoint after the stress is applied. A region of apoptosis, as a result of the treatment of the tissues/cells, is indicated where the signal amplitude rises by a significant factor, for example, where the signal amplitude rises by at least a factor of three, and more preferably by a factor of between three and six. The threshold of the increase in signal intensity that is used to indicate apoptosis in a particular biological system can be determined by correlating the change in signal intensity with a standard assay for apoptosis such as fluorescent staining of DNA when the biological system is exposed to a known apoptosis-inducing stress. A graph of the change in ultrasound signal intensity versus the percentage of apoptotic cells as measured using the standard apoptosis assay would then be the calibration curve used to determine the percentage of apoptotic cells in that biological system to any apoptosis-inducing stress using high frequency ultrasound imaging.
Subsequent to analyzing the signal amplitude data, a frequency analysis is performed on the radiofrequency ultrasound signals. This involves taking a Fourier transform of the data from both treated and untreated regions. At least 20 A-scan lines of radiofrequency data and preferably 20-50 lines are acquired from each region of interest and digitized by the ultrasound scanner. The window length over which the A-scan radiofrequency signal is digitized should correspond to between about 0.5 and 3 mm in the image. Ideally, the window length should be at the lower end of this range to reduce the effects of ultrasound attenuation in the frequency analysis. Fourier transforms of the acquired radiofrequency A-scan lines are calculated and then squared to give the Fourier power spectrum for each A-scan. The Fourier power spectrum is then normalized against a reference Fourier power spectrum of the ultrasound pulse from the transducer. This is achieved by dividing the Fourier power spectrum of the signals from the region of interest by the Fourier power spectrum of the ultrasound reflected from a hard surface such as a quartz flat. The normalized power spectra are calculated between bandwidth limits where the value of the reference Fourier power spectrum is xe2x88x9215 dB or 3% of the maximum value at the center frequency of the ultrasound imaging system. Linear regression is performed on the normalized power spectrum from each A-scan line. The linear regression lines fitted to each normalized Fourier power spectrum are then averaged over all the scan lines acquired from a region of interest to give an average fitted normalized Fourier power. The average fitted normalized Fourier power spectra are plotted as graphs of 10 log10 (normalized power) versus frequency. The slope of this line with function y=mx+c, is m. Apoptosis is indicated by the slope of the average fitted normalized Fourier power spectrum versus frequency becoming significantly more positive during treatment. An increase in the slope of at least 30% is indicative of apoptosis for cells in vitro. The increase in slope expected in tissues both ex-vivo and in-vivo varies with the particular tissue being examined. An increase in slope of at least 5% indicates apoptosis is occurring in tissues.
The method of frequency analysis described herein is well described in the literature and can be implemented readily by a person skilled in the art. The threshold of the increase in slope of the average fitted normalized Fourier power spectrum that is used in any particular biological system to indicate apoptosis can be determined by first correlating the increase in slope with a standard assay of apoptosis when a known apoptosis inducing agent is applied to that biological system. A graph of the change in slope of the average fitted normalized Fourier power spectrum versus percentage of apoptotic cells as measured using the standard apoptosis assay would then be the calibration curve used to determine the percentage of apoptotic cells in that biological system due to any apoptosis-inducing stress using high frequency ultrasound imaging.
Other methods in addition to our preferred method of Fourier analysis may be used to measure the change in the radiofrequency ultrasound signals due to apoptosis reflected back from the tissue or cells for example the increase in ultrasound signal due to apoptosis can be measured by calculating the mid-band fit of the average normalized Fourier power spectrum. The xe2x80x9cmid-band fitxe2x80x9d as referred to herein is defined as the value of the average normalized Fourier power spectrum at the center frequency of the chosen bandwidth. Similarly, the change in the frequency content of the ultrasound signals due to apoptosis can be measured by performing wavelet analysis, for example.
To further confirm the occurrence of apoptosis, the average scatterer size can be calculated from the radiofrequency spectra. This determination is not independent of slope calculated as set out above, and thus functions to verify apoptosis. Several methods have been published for calculating scatterer size from ultrasound backscatter signals including those of Lizzi et al (Theoretical framework for spectrum analysis in ultrasonic tissue characterization, Journal of the Acoustical Society of America 1983, 73, 1366-1373) and by Hall et al (Describing small-scale structure in random media using pulse echo ultrasound Journal of the Acoustical Society of America 1990, 87, 179-192; Parametric ultrasound imaging from backscatter coefficient measurements: Image formation and interpretation Ultrasonic Imaging 1990, 12, 245-267). Apoptosis is confirmed by a significant decrease in the average scatterer size in the region of interest. A decrease in scatterer size of between about 20-50% is generally indicative of apoptosis; however, decrease in scatterer size as calculated by these techniques is tissue dependent as well as dependent on the characteristics of the transducer used to calculate the theoretical curve of slope vs. scatterer radius (as shown in FIG. 7). Thus, a decrease in the scatterer size of at least 30% is more preferably indicative of apoptosis for cells in-vitro whereas a decrease in scatterer size of 20% is more preferably indicative of apoptosis for tissues both ex-vivo and in-vivo.
The threshold in decrease in scatterer size that is used to indicate apoptosis in any particular biological system can be determined by correlating the decrease in scatterer size calculated from the radiofrequency ultrasound data with the percentage of cells undergoing apoptosis as measured using a standard apoptosis assay when a known apoptosis-inducing agent is applied to that biological system. A graph of the decrease in scatterer size calculated from the ultrasound imaging radiofrequency data decrease versus percentage of apoptotic cells as measured using a standard assay would then be the calibration curve used to determine the percentage of apoptotic cells in that biological system due to any apoptosis-inducing stress using high frequency ultrasound imaging.
The present method may be more particularly characterized as follows:
1. Take ultrasound images (B-Scan or C-Scan) of the cells or tissues of interest before the apoptosis inducing treatment is applied.
2. Take a second set of images of the same area during and/or after treatment.
3. Calculate the signal level change in the region of interest. This can be achieved in two different ways: i) using a calibration curve from the ultrasound machine manufacturer to convert the final machine signal (pixel level) into an average radiofrequency signal power over the region of interest or ii) if the radiofrequency signal can be obtained from the machine, use this directly to calculate an average signal power over the region of interest.
4. The signal amplitude should be measured over a region of interest within the region of tissue exposed to the treatment, at any timepoint of interest after the treatment is applied and compared to the same region of interest in the images before the treatment was applied or compared to a neighboring area of untreated tissue.
5. An increase in signal power (intensity) by more than a factor of 9 (equivalent to an increase in signal amplitude by a factor of 3) over control or pretreatment measurements indicates that apoptosis is occurring in the treated region. In addition, the percentage of apoptotic cells in any biological system can be determined by using a calibration curve as set out above.
6. The second part of the process is to perform a frequency analysis on the radiofrequency data. The objective is to calculate the change in the slope of the average normalized Fourier power spectrum as set out above. Apoptosis is indicated if the change in slope is at least 30% for cells in vitro and at least 5% for tissues. In addition, the percentage of apoptotic cells in particular biological systems can be determined by using a calibration curve for each biological system as set out above.
7. A third calculation can be performed to confirm that the cause of changes in signal amplitude and frequency spectra slope is indeed apoptosis. This calculation derives the average scatterer size from the frequency spectra data. One method of calculating the average scatterer size is to employ the method of Lizzi et al. (supra). Apoptosis is confirmed if the average scatterer size decreases by about 20-50%, in addition to increases in the signal amplitude and the slope of the frequency spectra in the region of interest as set out above. In addition, the percentage of apoptotic cells in particular biological systems can be determined using a calibration curve for each biological system as set out above.
8. Data from the calculations above can be presented in several ways. Results of each of the individual calculations can be displayed and stored as a numeric value on the ultrasound imaging machine together with an indication as to whether they are consistent with apoptosis when compared to the threshold values set out above. Similarly, the percentage of apoptotic cells can be displayed and stored. In the preferred embodiment, increases in both signal intensity and slope of the average normalized Fourier power spectrum over the thresholds set out above would be required to report a positive finding that apoptosis is occurring in the sample as a result of the apoptosis-inducing stress.
It has been determined that the origin of the contrast in both signal amplitude and frequency spectrum in cells undergoing apoptosis is due to DNA condensation and fragmentation. This results in several ultrasound scattering DNA fragments being present in the apoptotic cell. Simulation studies show that this production of scattering particles in the cell during apoptosis should lead to a 3-6-fold increase in scatter amplitude signal and a 5% to 30% increase in the slope of the frequency spectrum, depending on the sample (i.e. cells vs. tissues).
The present invention has many potential applications. These include but are not restricted to human and veterinary, diagnostic and therapeutic applications. Such applications include the testing of drugs and other chemical compounds for toxicity mediated by apoptosis, the study of the effects of oncogenes and other genes on apoptosis and the measurement of the viability of organs and tissues for transplantation. The invention can be used and studied for these applications in cells in-vitro, in human and animal tissues ex-vivo and in-vivo and for specific clinical applications including the monitoring of patient responses to therapies including chemotherapy, radiation therapy, photodynamic therapy, gene therapy and any other therapy that may involve the triggering of an apoptotic response in cells. An example is to monitor apoptosis in inflammatory tissues after triggering of the immune system, which is a potential treatment for immune disorders. Further applications include studying the effects of ultraviolet, X-ray and gamma radiation on cells and tissues as well as chemical modifiers such as sunscreens aimed at protecting tissues from these radiations.
The present invention can be applied to several different ultrasound imaging methods. These methods include the use of external transducers that can be used to image tissues such as the skin and eye, invasive interstitial needle-based transducers that can be inserted directly into a target tissue deep within the body such as a tumour or normal tissue, intraluminal catheter-based transducers that are designed to image from within arteries for example and endoscopic or intracavitary ultrasound systems that can be used to image tissues including the easophagus and colon for example.